Estrogen binds to its receptors to regulate RNA transcription, stimulate cell proliferation and modulate metabolic signaling in many tissues during mammalian reproduction and development. Three genes for estrogenic binding proteins have been identified, encoding estrogen receptor (ER) α and β, and G-protein coupled estrogen receptor 1 (GPER1). ER-α and β have similar structural and functional domains, containing activation function domain 1 (AF-1), a DNA binding domain (DBD), a dimerization domain and activation function domain 2 (AF-2), which is the ligand binding domain (LBD). They both belong to the nuclear super family of ligand-dependent transcription factors and have highly conserved DBD and LBD regions (95%). They regulate RNA transcription upon ligand binding, which results in ligand-receptor complexes that can dimerize and translocate into the nucleus, where they bind to estrogen response elements (EREs) found in the promoters of estrogen-responsive genes. This type of modulation is typically referred to as the classical estrogen pathway. ER-α and β also regulate diverse biological functions through membrane-initiated estrogen signaling (MIES), associating with plasma membrane by interaction with their ligand binding domain. The detailed molecular mechanisms of signaling by membrane-associated ERs are still unclear. The modulatory effects of estrogen mediated by membrane-associated receptors on cell proliferation, matrix/migration, metabolism and glucose homeostasis have been reviewed (1, 2). Furthermore, studies on ER knockout mice indicate that ER-α is the dominant functional estrogen receptor, as compared to ER-β. Three transcription variants of ER-α, -66, -46 and -36, have been found. ER-α-36 lacks the AF-1 domain and contains a partial ligand binding domain. It has been found localized to the cell membrane and cytosol. Since ER-α-36 is restricted to modulating MIES and was found to be uniquely expressed in tamoxifen-resisted cancer cells, such as MDA-MB-231 and Hec1A, MIES modulated by membrane-associated ER is thought to be responsible for the resistance to anti-estrogen therapy found by some researchers (3,4).
Orphan G-protein coupled receptor 30 (GPER1) was found to bind E2 (17-beta estradiol, an estrogen) (5) and modulate cell proliferation, resulting in resistance to anti-estrogen therapy. However, its physiological function is still a matter of controversy among some investigators (3). GPER1 knockout mice have been shown to exhibit cardiovascular and metabolic defects, with no apparent effect on fertility (6). Thus GPER1 may be involved in the modulation of estrogen-mediated metabolic signaling.
The decreased production of estrogen in postmenopausal women leads to symptoms that may adversely affect their quality of life for decades. Hormone (estrogen) replacement therapy (HRT/ERT) has been utilized to treat these symptoms since the 1940s. Studies showing an increased risk of breast and uterine cancer, as well as thromboembolism morbidity, associated with HRT have lead to a recent decline in its usage, and postmenopausal symptoms remain a problem for many older women. Selective estrogen receptor modulators (SERMs) have been utilized as treatments to regulate estrogen signaling since the 1990s. However, the lack of a more complete understanding of the molecular mechanisms involved and interfering cross-talk between selective modulators with different estrogen receptors have made it difficult to design treatment regimens that avoid the development of drug resistance and serious side effects during clinical usage.
Tamoxifen was marketed as an antagonist of the estrogen classical pathway to treat breast cancer patients, and was also reported as an agonist of ESR-α-36, potentially leading to anti-estrogen therapy resistance while stimulating the growth of endometrial epithelium cells, resulting in endometrium cancer (7, 8). Raloxifene was marketed as an upgraded version of tamoxifen, having fewer side effects and the advantages of inhibiting cancer cell migration and preventing postmenopausal symptoms, such as osteoporosis. However, raloxifene can still cause serious side effects common to tamoxifen treatment, such as thromboembolism and non-alcohol steatohepatitis (NASH) (9, 10). The detailed mechanisms responsible for the side effects caused by either raloxifene or tamoxifen are still unclear. Ipriflavone is a derivative of phytohormone, and its metabolite binds to the ER-α LBD with a lower affinity than E2, exhibiting reduced estrogenic effects. The metabolites of ipriflavone and isoflavone show comparable binding affinity and activity with ER-β as E2, and they have been utilized in some countries as a medicine to prevent osteoporosis. However, their effectiveness was not supported in at least one clinical trial (11). Moreover, potential side effects as seen with traditional HRT are still a concern to some investigators (12).
2β,7α-diethyl-A-nor-5α-androstane-2α,17β-diol (anordiol) was first reported as possessing anti-estrogenic activity by Pincus et al in the 1960s (13, 14). Li, R. L. esterified anordiol using propionic acid to synthesize 2α,17α-diethynyl-A-nor-5α-androstane-2β,17β-diol dipropionate (anordrin, ANO) in 1969. Anordrin was marketed as an antifertility medicine using the brand name AF-53 in China beginning in 1976. Estrogen is known to cause hormone-induced cancer, and anordrin, as an estrogen receptor antagonist, was subsequently found to inhibit malignant cell growth (15, 16, 17). As a non-prescription medicine in China, Chinese physicians used it as an anti-tumor agent for nearly a decade under legally licensed conditions. However, confusing results were reported for many patients during clinical therapy. Its clinical usage as an anti-tumor agent was stopped in 1998 after the introduction of the clinical trial law in China, and all of the relevant clinical data were never collected and studied.
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